Polymorphonuclear leukocytes (neutrophils or PMNs) and mononuclear phagocytes (monocytes) play an important part in tissue injury, infection, acute and chronic inflammation and wound healing. There is some indication that a number of chronic diseases are caused by pathological proteolysis due to overstimulation of the PMNs. Such overstimulation may be caused by, for instance, autoimmune response, chronic infection, tobacco smoke or other irritants, etc.
PMNs migrate from the blood to the site of inflammation and, following appropriate stimulation, they release oxidant compounds (O.sub.2., O.sub.2.sup.-, H.sub.2 O.sub.2 and HOCl ) as well as granules containing a variety of proteolytic enzymes. The secretory granules contain, inter alia, alkaline phosphatase; metalloproteinases such as gelatinase and collagenase; and serine proteases such as neutrophil elastase, cathepsin G and proteinase-3.
Latent metalloproteinases are released together with tissue inhibitor of metalloproteinase (TIMP). The activation mechanism has not been fully elucidated, but it is likely that oxidation of thiol groups and/or proteolysis play a part in the process. Also, free metalloproteinase activity is dependent on inactivation of TIMP.
PMNs contain large quantities of serine proteases, and about 200 mg of each of the leukocyte proteases are released daily to deal with invasive agents in the body. Acute inflammation leads to a many-fold increase in the amount of enzyme released. In the azurophil granules of polymorphonuclear leukocytes, the serine proteases neutrophil elastase, cathepsin G and proteinase-3 are packed as active enzymes complexed with glucosaminoglycans. These complexes are inactive but dissociate on secretion to release the active enzymes. Under normal conditions, proteolysis is kept at an acceptably low level by large amounts of the inhibitors .alpha..sub.1 -proteinase inhibitor (.alpha..sub.1 -PI), .alpha..sub.1 -chymotrypsin inhibitor (.alpha..sub.1 -ChI) and .alpha..sub.2 macroglobulin that are found in plasma. However, the PMNs are able to inactivate the inhibitors locally. Thus, .alpha..sub.1 -PI, which is the most important inhibitor of neutrophil elastase, is sensitive to oxidation at the reactive center (Met-358) by oxygen metabolites produced by stimulated PMNs. This reduces the affinity of .alpha..sub.1 -PI for neutrophil elastase by approximately 2000 times.
After local neutralization of .alpha..sub.1 -PI, the neutrophil elastase is able to degrade a number of inhibitors of other proteolytic enzymes. Neutrophil elastase cleaves .alpha..sub.1 -ChI and thereby promotes cathepsin G activity. It also cleaves TIMP, resulting in tissue degradation by metalloproteinases. Furthermore, neutrophil elastase cleaves antithrombin III, heparin cofactor II, and tissue factor pathway inhibitor (TFPI), which probably promotes clot formation. On the other hand, the ability of neutrophil elastase to degrade coagulation factors is assumed to have the opposite effect, such that the total effect of neutrophil elastase is unclear. The effect of neutrophil elastase on fibrinolysis is less ambiguous. Fibrinolytic activity increases when neutrophil elastase cleaves plasminogen activator inhibitor and .alpha..sub.2 plasmin inhibitor. Furthermore, both of these inhibitors are oxidized and inactivated by O.sub.2 metabolites.
Protein inhibitors are classified into a series of families based on extensive sequence homologies among the family members and the conservation of intrachain disulfide bridges (for review, see Laskowski and Kato, Ann. Rev. Biochem. 49: 593-626, 1980). Serine protease inhibitors of the Kunitz family are characterized by their homology with aprotinin (bovine pancreatic trypsin inhibitor). Aprotinin is known to inhibit various serine proteases including trypsin, chymotrypsin, plasmin and kallikrein. Kunitz-type inhibitor domains have been reported in larger proteins such as the inter-.alpha.-trypsin inhibitors (Hochstrasser et al., Hoppe-Seylers Z. Physiol. Chem. 357: 1659-1661, 1969 and Tschesche et al., Eur. J. Biochem. 16: 187-198, 1970) and the .beta.-amyloid protein precursor. The .beta.-amyloid protein precursor (APP) contains an approximately 40 amino acid fragment that forms the senile plaques observed in Alzheimer's patients, patients with Down's syndrome and in aged normal patients. The gene encoding APP yields three alternatively spliced mRNAs, two of which have been demonstrated to encode Kunitz-type inhibitor domains (see Ponte et al., Nature 331: 525-528, 1988; Tanzi et al., Nature 331: 528- 530, 1988 and Kitaguchi et al., Nature 331: 530-532, 1988). In addition to the Kunitz-type inhibitor domain, each protein precursor contains a signal peptide, a cysteine-rich region, a highly negatively charged region, a transmembrane domain and an intracellular domain (see Kitaguchi et al. ibid.).
Of the Kunitz-type inhibitors, aprotinin is used therapeutically in the treatment of acute pancreatitis, various states of shock syndrome, hyperfibrinolytic hemorrhage and myocardial infarction (see, for example, Trapnell et al., Brit. J. Surg. 61: 177, 1974; McMichan et al., Circulatory shock 9: 107, 1982; Auer et al., Acta Neurochir. 49: 207, 1979; Sher, Am. J. Obstet. Gynecol. 129: 164, 1977; and Schneider, Artzneim.-Forsch. 26: 1606, 1976). Administration of aprotinin in high doses significantly reduces blood loss in connection with cardiac surgery, including cardiopulmonary bypass operations (see, for example, Bidstrup et al., J. Thorac. Cardiovasc. Surg. 97: 364-372, 1989; van Oeveren et al., Ann. Thorac. Surg. 44: 640-645, 1987). It has previously been demonstrated (Wenzel and Tschesche, Angew. Chem. Internat. Ed. 20: 295, 1981) that certain aprotinin analogs, e.g. aprotinin (1-58, Val15), exhibit a relatively high selectivity for granulocyte elastase and an inhibitory effect on collagenase. Aprotinin (1-58, Ala15) has a weak effect on elastase, while aprotinin (3-58, Arg15, Ala17, Ser42) exhibits an excellent plasma kallikrein inhibitory effect (WO 89/10374).
However, when administered in vivo, aprotinin has been found to have a nephrotoxic effect in rats, rabbits and dogs after repeated injections of relatively high doses (Bayer, Trasylol, Inhibitor of Proteinase; Glaser et al. in "Verhandlungen der Deutschen Gesellschaft fur Innere Medizin, 78. Kongress," Bergmann, Munich, 1972, pp. 1612-1614). The nephrotoxicity (appearing, i.e., in the form of lesions) observed for aprotinin might be ascribed to the accumulation of aprotinin in the proximal tubulus cells of the kidneys as a result of the high positive net charge of aprotinin, which causes it to be bound to the negatively charged surfaces of the tubuli. This nephrotoxicity makes aprotinin less suitable for clinical purposes, particularly in those uses requiring administration of large doses of the inhibitor (such as cardiopulmonary bypass operations). Furthermore, aprotinin is a bovine protein, which may induce an immune response upon administration to humans.
It is therefore an object of the present invention to provide novel human protease inhibitors of the Kunitz family of inhibitors with similar inhibitor profiles for use in the treatment of acute pancreatitis, various states of shock syndrome, hyperfibrinolytic hemorrhage and myocardial infarction. It is further an object of the present invention to provide novel amyloid protein precursor homologues for use in studying the relative levels of the precursor in patients exhibiting Alzheimer's disease and to identify patients with mutations in the protein precursor.